PROJECT SUMMARY Advances in cancer immunotherapy have great potential for treating tumors that are refractory to conventional treatments, and T cells primed ex vivo by natural or artificial antigen-presenting cells (APCs) to target and kill cancer cells have been shown clinically to improve survival in patients with highly aggressive cancers. APCs normally prime T cells by presenting a tumor antigen-specific signal 1, consisting of a major histocompatibility complex (MHC) I molecule with a tumor antigen peptide; a co-stimulatory signal 2 that directs the action of the T cells upon recognition of the tumor; and a secreted signal 3 for recruitment and activation of immune cells. Instead of engineering the patient's APCs to direct a T-cell response against a tumor or fabricating artificial, synthetic APCs, both of which are costly, complex, and/or patient-specific processes, we propose to reprogram cancer cells themselves to become tumor-derived APCs (tAPCs). Because tumor cells already intrinsically express signal 1 (tumor antigen in the context of MHC I), they can be engineered in situ to express the other necessary signals and therefore act as APCs, directing cytotoxic T-cell responses against themselves. Tumor cells with low MHC I expression will stimulate natural killer (NK) cells to aid this purpose. We have designed synthetic, non-viral nanoparticles that can deliver DNA to cancer cells with high efficacy and specificity over healthy tissue, and we will inject these into a tumor mass to induce expression of signal 2 and signal 3, using two different in vivo orthotopic tumor models (melanoma and triple-negative breast cancer) and four in vitro tumor models as examples. Our proposed experiments will first optimize the nanoparticle formulation for high expression and cancer specificity in vitro and in vivo. We will then demonstrate activation and T and NK cells after reprogramming of cancer cells to express signals 2 and 3 along with signal 1 both in vitro and in vivo. Finally, we will show anti-cancer efficacy after intratumoral injection of these nanoparticles in immunocompetent murine cancer models, with a focus on models of metastasis, and examine the immunological mechanisms underlying our technology. Importantly, because our goal is to use DNA-delivery nanoparticles to stimulate the immune system to kill cancer cells, rather than directly killing the melanoma cells by gene transfer, our strategy requires only a representative subset of malignant cells to be successfully transfected. If successful, this could result in an affordable, fully synthetic, local, antigen-agnostic therapy that nevertheless leads to antigen-specific systemic immune rejection of tumors.